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miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses

  • Aleksandra Smoczynska
  • Pawel Sega
  • Agata Stepien
  • Katarzyna Knop
  • Artur Jarmolowski
  • Andrzej PacakEmail author
  • Zofia Szweykowska-KulinskaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan® technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR® Green is not used but the more sensitive TaqMan® probe that on 5′ end contains a covalently attached fluorophore and on 3′ quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.

Key words

microRNA Stem-loop primer RT-qPCR RT-ddPCR TaqMan Probes 

Notes

Acknowledgment

The project is supported by the National Science Centre, Poland based on the decisions number DEC-2013/11/B/NZ9/01761, UMO-2015/19/N/NZ9/00218, 2013/10/A/NZ1/00557, UMO-2016/23/N/NZ1/00005, UMO-2016/21/B/NZ9/00550, UMO-2016/23/B/NZ9/00857 and UMO-2016/23/B/NZ9/00862, by the Foundation for Polish Science (grants START 2017 to Agata S. and Katarzyna K.), and by KNOW RNA Research Centre in Poznań (No. 01/KNOW2/2014).

References

  1. 1.
    Pacak A, Barciszewska-Pacak M, Swida-Barteczka A, Kruszka K, Sega P, Milanowska K, Jakobsen I, Jarmolowski A, Szweykowska-Kulinska Z (2016) Heat stress affects Pi-related genes expression and inorganic phosphate deposition/accumulation in barley. Front Plant Sci 7:926CrossRefGoogle Scholar
  2. 2.
    Barciszewska-Pacak M, Milanowska K, Knop K, Bielewicz D, Nuc P, Plewka P, Pacak AM, Vazquez F, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2015) Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses. Front Plant Sci 6:410CrossRefGoogle Scholar
  3. 3.
    Kruszka K, Pacak A, Swida-Barteczka A, Nuc P, Alaba S, Wroblewska Z, Karlowski W, Jarmolowski A, Szweykowska-Kulinska Z (2014) Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley. J Exp Bot 65:6123–6135CrossRefGoogle Scholar
  4. 4.
    Schommer C, Bresso EG, Spinelli SV, Palatnik JF (2012) Role of microRNA miR319 in plant development. In: MicroRNAs in plant development and stress responses. Springer, New York, pp 29–47CrossRefGoogle Scholar
  5. 5.
    Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263CrossRefGoogle Scholar
  6. 6.
    Wang JW, Wang LJ, Mao YB, Cai WJ, Xue HW, Chen XY (2005) Control of root cap formation by MicroRNA-targeted auxin response factors in Arabidopsis. Plant Cell 17:2204–2216CrossRefGoogle Scholar
  7. 7.
    Bari R, Datt Pant B, Stitt M, Scheible WR (2006) PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiol 141:988–999CrossRefGoogle Scholar
  8. 8.
    Pant BD, Buhtz A, Kehr J, Scheible WR (2008) MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J 53:731–738CrossRefGoogle Scholar
  9. 9.
    Xu F, Liu Q, Chen L, Kuang J, Walk T, Wang J, Liao H (2013) Genome-wide identification of soybean microRNAs and their targets reveals their organ-specificity and responses to phosphate starvation. BMC Genomics 14:66CrossRefGoogle Scholar
  10. 10.
    Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077–1084CrossRefGoogle Scholar
  11. 11.
    Wang X, Tong Y, Wang S (2010) Rapid and accurate detection of plant miRNAs by liquid northern hybridization. Int J Mol Sci 11:3138–3148CrossRefGoogle Scholar
  12. 12.
    Várallyay É, Burgyán J, Havelda Z (2007) Detection of microRNAs by Northern blot analyses using LNA probes. Methods 43:140–145CrossRefGoogle Scholar
  13. 13.
    Válóczi A, Hornyik C, Varga N, Burgyán J, Kauppinen S, Havelda Z (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32:e175–e175CrossRefGoogle Scholar
  14. 14.
    Yao X, Huang H, Xu L (2012) In situ detection of mature miRNAs in plants using LNA-modified DNA probes. Methods Mol Biol 883:143–154CrossRefGoogle Scholar
  15. 15.
    Javelle M, Timmermans MC (2012) In situ localization of small RNAs in plants by using LNA probes. Nat Protoc 7:533–541CrossRefGoogle Scholar
  16. 16.
    Niedojadło J, Dełeńko K, Niedojadło K (2016) Regulation of poly (A) RNA retention in the nucleus as a survival strategy of plants during hypoxia. RNA Biol 13:531–543CrossRefGoogle Scholar
  17. 17.
    Yang X, Li L (2012) Analyzing the microRNA transcriptome in plants using deep sequencing data. Biology 1:297–310CrossRefGoogle Scholar
  18. 18.
    Gunaratne PH, Coarfa C, Soibam B, Tandon A (2012) miRNA data analysis: next-gen sequencing. Methods Mol Biol 822:273–288CrossRefGoogle Scholar
  19. 19.
    Motameny S, Wolters S, Nürnberg P, Schumacher B (2010) Next generation sequencing of miRNAs–strategies, resources and methods. Genes 1:70–84CrossRefGoogle Scholar
  20. 20.
    Shi R, Sun YH, Zhang XH, Chiang VL (2012) Poly(T) adaptor RT-PCR. Methods Mol Biol 822:53–66CrossRefGoogle Scholar
  21. 21.
    Prigge MJ, Wagner DR (2001) The Arabidopsis serrate gene encodes a zinc-finger protein required for normal shoot development. Plant Cell 13:1263–1279CrossRefGoogle Scholar
  22. 22.
    Grigg SP, Canales C, Hay A, Tsiantis M (2005) SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis. Nature 437:1022–1026CrossRefGoogle Scholar
  23. 23.
    Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17CrossRefGoogle Scholar
  24. 24.
    Zielezinski A, Dolata J, Alaba S, Kruszka K, Pacak A, Swida-Barteczka A, Knop K, Stepien A, Bielewicz D, Pietrykowska H (2015) mirEX 2.0-an integrated environment for expression profiling of plant microRNAs. BMC Plant Biol 15:144CrossRefGoogle Scholar
  25. 25.
    Knop K, Stepien A, Barciszewska-Pacak M, Taube M, Bielewicz D, Michalak M, Borst JW, Jarmolowski A, Szweykowska-Kulinska Z (2017) Active 5΄ splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes. Nucleic Acids Res 45:2757–2775Google Scholar
  26. 26.
    Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–8610CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Aleksandra Smoczynska
    • 1
  • Pawel Sega
    • 1
  • Agata Stepien
    • 1
  • Katarzyna Knop
    • 1
  • Artur Jarmolowski
    • 1
  • Andrzej Pacak
    • 1
    Email author
  • Zofia Szweykowska-Kulinska
    • 1
    Email author
  1. 1.Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of BiologyAdam Mickiewicz University, PoznańPoznańPoland

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